Triterpenoid Dammarans From The Bark of Aglaia Smithii (Meliaceae) And Their Toxic Activity Against Artemia Salina And Cytotoxic Activity Against Murine Leukimia Cells P-388.

(1)

TRITERPENOID DAMMARANS FROM THE BARK OF

AGLAIA SMITHII

(MELIACEAE) AND THEIR TOXIC ACTIVITY AGAINST ARTEMIA SALINA

AND CYTOTOXIC ACTIVITY AGAINST MURINE LEUKEMIA CELLS P-388

Desi Harneti Putri Huspaa), Unang Supratman a), Tati Herlina a), Syafruddin b) a)

Jurusan Kimia FMIPA UNPAD, b) Lembaga Eijkman Jakarta

* Corresponding author, tel/fax : 022-7794391, email: [email protected]

ABSTRACT

Aglaia smithii is a higher plant belonging to Meliaceae family and widely distributed in South East Asia. Plants of Meliaceae family were known as a source of cytotoxic (anticancer) substances. The methanolic extract from the dried bark of A. smithii was concentrated and extracted successively with n-hexane, methylene chloride and ethyl acetate. By using Brine Shrimp Lethality Test (BSLT) to follow the separation (bioassay guided isolation), the compounds of n-hexane extract was separated by combination of column and thin layer chromatography to yield two toxic compounds. The chemical structure of active compounds were determined by spectroscopic data and compared with those spectra data reported previously were identified as eichlerianic acid (1) and aglinin A (2). The toxic activity of compounds (1) and (2) against brine shrimp (A. salina) were evaluated by Meyer method and showed activity with LC50 24,7and

36,4; µg/mL, respectively. The cytotoxic activity of compounds (1) and (2) against murine leukemia cells P-388 were evaluated by Alley Method and showed weak activity with IC50 34 and 42 µg/mL, respectively.

Keywords:Aglaia smithii; Meliaceae; brine shrimp lethality test; murine leukemia cell P-388; triterpenoid dammaran

INTRODUCTION

Plants belonging to the genus

Aglaia are a rich source of tetracyclic

triterpenoids of the cycloartane,

dammarane and tirucallane series [1]

In continuation of our work on plants

belonging to this genus collected in

Indonesia, we have isolated from the

bark of Aglaia smithii the known

dammaran triterpenoids eichlerianic acid

(1) and aglinin A (2).

Aglaia smithii is a higher plant

belonging to Meliaceae family and

(2)

Plants of Meliaceae family were

known as a source of cytotoxic

(anticancer) substances [2-4] . The

methanol extract from bark of A.

smithii showed significant activity

against brine shrimp (Artemia salina)

and murine leukemia cells P-388. This

plant has not yet been subjected to

any phytochemical or biological

investigation. The methanolic extract

from the dried bark of A. smithii was

concentrated and extracted

successively with n-hexane,

methylene chloride and ethyl acetate.

By using Brine Shrimp Lethality Test

(BSLT) to follow the separation

(bioassay guided isolation), the

compounds of n-hexane extract was

separated by combination of column

and thin layer chromatography to yield

two toxic compounds. The toxic

activity of compounds (1) and (2)

against brine shrimp (A. salina) were

evaluated by Meyer method and

showed activity with LC50 24,7 and

36,4; µg/mL, respectively. The

cytotoxic activity of compounds (1)

and (2) against murine leukemia cells

P-388 were evaluated by Alley Method

and showed weak activity with IC50 34

and 42 µg/mL, respectively.

EXPERIMENTAL SECTION Materials

Bark material of Aglaia smithii Corr.

was collected in Bogor Botanical

Garden West Java Indonesia, during

October 2006. The specimen was

identified and deposited at the

Herbarium Bogoriense West Java

Indonesia.

Instrumentation

Rotavapor R-200 Buchi with vacum Vac

V-500 Buchi. Fischer-Johns Melting

Point Apparatus. Spektrofotometer FTIR

Shimadzu 8400 and FTIR Spectrum

One Perkin Elmer. Spektrofotometer

NMR (Nuclear Magnetic Resonance)

JEOL JNM ECA-500. Mariner

Biospectrometry, Hitachi L 6200, sistem

ESI (Electrospray Ionisation), positive

ion mode and Shimadzu LCMS solution,

negative ion mode. Bruker SMART

APEX Diffractometer.

(3)

Extraction and isolation

Dried bark of A. smithii (3 kg)

were extracted exhaustively with

methanol at room temperature. The

extract (286 g) was diluted with

methanol : water (8:2 v/v) and

partitioned with n-hexane. The extract

(13 g) was repeatedly

chromatographed on silica gel yielding

eiclerianic acid (23 mg) (1)

n-hexane/ethyl acetate, 7 : 3, (2) n

-heksana/CH2Cl2/MeOH, 1,4 : 85 : 0,1.

Aglinin A (21 mg) CH2Cl2/MeOH,

MeOH : H2O, 9:1.

Bioassay

The toxic activity of compounds (1)

and (2) against brine shrimp (A. salina) were evaluated by Meyer

method and showed activity with LC50

24,7and 36,4; µg/mL, respectively.

The cytotoxic activity of compounds

(1) and (2) against murine leukemia cells P-388 were evaluated by Alley

Method and showed weak activity with

IC50 34 and 42 µg/mL, respectively.

RESULTS AND DISCUSSION

The 3,4 secodammaranes, eichlerianic

acid (1) and aglinin A (2) were isolated

from the n-hexane extract of A. smithtii

bark by chromatography on silica gel.

They have been found previously in A.

lawii leaves [5]. However, they have

been described in other genera of the

family Meliaceae and were identified on

the basis of comparison of their spectral

data with literature values [1,5]. They all

possess the same

20S,24-epoxy-25-hydroxy chain at C-17.

Eichlerianic acid

Eichlerianic acid showed an

[M-H]- peak at m/z 473 in the LC-MS,

coresponding to molecular formula

C30H50O4.

Recent detailed NMR studies have

unambiguously demonstrated that the

24R and 24S isomer can be easily

distinguished by the resonances of C-24

(δC 83.2 for the R-isomer and δC 86.5

for the S-isomer) [5]. The chemical shifts

and coupling patterns of the H-24 differ

also from each other (δH3.7, J =7 and 7

Hz and δH 3.6, J = 10 and 5.5 Hz,

(4)

carboxyl group at C-3 and double

bond at C-4 and C-28 with HMBC

correlation H-28/C-5, C-29;

H-29/C-28,C-5,C-4; H-2/C-3 indicated ring A is

open.

The IR showed the absorption of a

carbonyl group at 1708 cm-1.

In the 13C NMR the signal of the

carbonyl appears at δ 179.7

suggesting a carboxylic acid residue.

The1H NMR exhibited the signal of

seven tertiary methyls between δ 0.8

and 1.3, ten methylene, four methin

and of an oxymethineat δ 3.63 typical

of the H-24 in

20S,24S-epoxy-25-hydroxydammaranes. All the 13C

signals were identic to the ones of

eichlerianic acid.

Aglinin A

Aglinin A showed an [M-H]- peak

at m/z 489,3 in the LC-MS,

coresponding to molecular formula

C

30

H

50

O

5.

The IR spectrum showed the

absorption of a carboxyl at 1710 cm-1.

In the 13C NMR spectrum (Table 2),

the signal of the C-3 carbonyl appeared

at δ 178.5.

These data as well as all the signals of

the dammarane fused ring moiety were

similar to the ones of the

3,4secodammaranes eichleirianic acid

previously isolated. The side chain

signals, however, differed from those of

the latter. In the NMR spectra, the

typical signals of the C-24 oxymethine

were absent. Instead, a signal of a

C-OH at low field (δ 108.7) was observed

in the 13C NMR suggesting that the

tetrahydrofuran ring was replaced by a

five membered hemiacetal ring. The 1D

and 2D (COSY, HMQC, HMBC) NMR

data were in agreement with the

structure depicted in Figure 1, especially

the diagnostic HMBC correlations

21/C-17, C-20,C-22 and 26,

Me-27/C-24. C-20 was assigned the S

configuration similar to most of the

dammarane triterpenes particularly

those isolated from the Aglaia genus

(5)

Two toxic 3,4-secodammarane

triterpenes, eichlerianic acid (1) and aglinin A (2), were isolated from the stem bark of Aglaia smithii with

bioactivity (Brine Shrimp Lethality

Test) guided chromatografic

fractionation .

Education, Directorate of Higher

Education, Republik Indonesia, for

financial support (STRANAS Project

2010) and LIPI Serpong for NMR

dammarane triterpenes from

Aglaia foveolata. Phytochemistry

49(6): 1745-1748.

2. Omar S., Zhang, J., Kinnon,

M.S., Leaman, D., Arnason, J.T.,

and Philogen, B.J.R. 2003.

Traditionally-used antimalarials

from Meliaceae. Current Top Medicinal Chemistry 3(2): 133-139.

3. Nugroho, B. W., Edrada, R.A.,

Wray, V., Witte L., Bringmann,

G., Gehling, M., and Proksch, P.

1999. An insectisidal

rocaglamida derivates and

related compounds from Aglaia odorata (Meliaceae).

Phytochemistry 51: 367-376. 4. Nakatani, M., Abdelgalell,

S.A.M., Sand, M.M.G., Huang,

R.C., Doe, N., and Iwagawa, T.

2004. Phragmalin Limonoids

from Chukrasia tabularus.

Phytochemistry65: 2833-2841. 5. Mohammad, K.,Martin, M.-T.,

Leroy, E., Tempete, C., Sevenet,

T., Awang, K., and Pais, M.

1999. Dammarane Triterpenes

and Pregnane Steroids from

Aglaia lawii and A. tomentosa.

(6)

Table 1 13C (125 MHz) and 1H NMR (500 MHz) data for eichlerianic acid (CDCl3)

Posisi C C-NMR.

c (mult.)

1

H-NMR

H, (H, mult., J (Hz))

HMBC (HC)

1 34,44 (t) 1,43 (2H,m)

2 28,14 (t) 2,20; 2,39 (2H,m) C-3

3 178,39 (s) -

4 147,70 (s) -

5 49,98 (d) 1,96 (1H,m)

6 25,00 (t) 1,87 (2H,m)

7 34,06 (t) 1,58 (2H, m) C-5, C-9

8 40,22 (s) -

9 41,36 (d) 1,52 (1H,m)

10 39,25 (s) -

11 22,52 (t) 1,43 (2H, m)

12 24,76 (t) 1,38 (2H, m)

13 43,07 (d) 1,64 (1H, m)

14 50,55 (s) -

15 31,65 (t) 1,46 (2H, m)

16 27,08 (t) 1,34 (2H, m)

17 49,90 (d) 1,86 (1H, m)

18 16,50 (q) 0,89 (3H, s) C-7,C-8, C-14

19 20,39 (q) 0,86 (3H, s) C-1, C-5,C-9, C-10

20 86,73 (s) -

21 27,40 (q) 1,14 (3H, s) C-17,C-20,C-22

22 34,87 (t) 1,58 (2H, m)

23 26,51 (t) 1,62 (2H, m)

24 86,52 (d) 3,63 (1H, dd, J = 5,5 dan 9,8)

25 70,44 (s) -

26 28,08 (q) 1,19 (3H, s) C-27,C-25,C-24

27 24,23 (q) 1,11 (3H, s) C-26, C-25,C-24

28 113,63 (t) 4,66; 4,85 (2H, brs) C-29,C-5

29 23,38 (q) 1,73 (3H, s) C-5,C-28,C-4

(7)

Table 2 13C (125 MHz) and 1H NMR (500 MHz) data for aglinin A (CDCl3)

Posisi C C-NMR.

c (mult.)

1

H-NMR

H, (H, mult., J (Hz))

HMBC

1 34,08 (t) 1,19; 1,23 (2H,m)

2 28,22 (t) 2,15; 1,40 (2H,m) C-1, C-3

3 178,54 (s) -

4 147,75 (s) -

5 50,95 (d) 1,95 (1H,m)

6 27,98 (t) 1,82; 1,96 (2H,m)

7 31,24 (t) 1,08 (2H, m)

8 39,17 (s) -

9 43,33 (d) 1,52 (1H,m)

10 40,24 (s) -

11 22,20 (t) 1,38; 1,43 (2H, m)

12 27,58 (t) 1,82; 1,96 (2H, m)

13 41,42 (d) 1,49 (1H, m)

14 50,51 (s) -

15 34,33 (t) 1,60 (2H, m) C-8, C-14

16 26,35 (t) 1,36 (2H, m)

17 51,05 (d) 1,99 (1H, m) C-21

18 20,37 (q) 0,88 (3H, s) C-7,C-8, C-9,C-14

19 15,52 (q) 1,00 (3H, s) C-1, C-5, C-10

20 89,15 (s) -

21 24,71 (q) 1,13 (3H, s) C-17,C-20,C-22

22 31,78 (t) 1,07; 1,84 (2H, m) C-20

23 31,82 (t) 1,82 (2H, m)

24 108,74 (s) -

25 74,76 (s) -

26 24,78 (q) 1,28 (3H, s) C-24, C-25

27 24,32 (q) 1,25 (3H, s) C-24, C-25, C-27

28 113,67 (t) 4,65; 4,85 (2H, brs) C-29,C-5

29 23,36 (q) 1,71 (3H, s) C-5,C-28,C-4

(8)

ARTIKEL ILMIAH

HIBAH PENELITIAN STRATEGI NASIONAL

TRITERPENOID YANG BERSIFAT ANTIMALARIA DARI

TUMBUHAN MELIACEAE INDONESIA

Oleh :

Dr. Desi Harneti Putri Huspa

Prof. Dr. Unang Supratman

dr. Syafruddin, Ph.D

Dr. Tati Herlina

DIBIAYAI OLEH :

DIREKTORAT JENDRAL PENDIDIKAN TINGGI, KEMENTRIAN PENDIDIKAN NASIONAL

SESUAI DENGAN SURAT PERJANJIAN PELAKSANAAN HIBAH PENELITIAN

NOMOR : 153/SP2H/PP/DP2M/III/2010 TANGGAL 1 MARET 2010

Triterpenoid Dammarans From The Bark of Aglaia Smithii (Meliaceae) And Their Toxic Activity Against Artemia Salina And Cytotoxic Activity Against Murine Leukimia Cells P-388.